Serum amyloid A (SAA) refers to a family of highly conserved proteins of unknown function that circulate in association with high density lipoproteins (HDL). The two best characterized SAA isotypes are hyperinduced in response to tissue injury. Patients with inflammatory disorders such rheumatoid arthritis and ankylosing spondylitis have chronically elevated levels of SAA and are predisposed to reactive amyloidosis. In this fatal disease, tissue function is compromised by fibrillar deposits of amyloid A (AA) protein, a peptide most commonly containing the amino-terminal 76 residues of 104-amino acid SAA. The overall goal of this project is to understand the metabolism of SAA and to relate metabolic processes to amyloidogenesis. Ultimately, it is hoped that the biochemical and cellular interactions involved in SAA metabolism may direct us to the function of this protein. It seems likely that the function served by SAA during the acute phase response is in some way related to its association with HDL. This interaction also appears to protect SAA from degradation. Based on preliminary studies, it is hypothesized that under normal circumstances SAA degradation occurs in an acidic environment and is initiated by a cathepsin D-like enzyme. This protease cleaves SAA in the amino-terminal portion thereby removing the region not only essential for lipid binding, but also that part required for the intermolecular associations initiating fibrillogenesis. Experiments have been designed to begin to experimentally test the proposed metabolic scheme: (1) The HDL-binding domain of SAA will be identified. Recombinant SAA (rSAA) proteins having either truncated amino-termini or amino acid substitutions will be evaluated in terms of HDL-binding capability. (2) Plasma half-lives of murine and human SAAs will be determined to gain insight into the clearance/degradative mechanism of SAA. The possibility that cathepsin D plays a physiologic role in SAA catabolism will be tested in pepstatin-treated mice. In vivo treatment with this inhibitor has been shown to result in prolonged plasma half-lives of cathepsin D-degraded proteins. (3) The involvement of lysosomal proteases in SAA degradation and amyloidogenesis will be characterized both in vitro and in vivo. In vivo experiments will include investigating the biodistribution and possible lysosomal presence of SAA, assessing the effects of impaired cathepsin activity on SAA metabolism and amyloidogenesis, and determining cathepsin mRNA and activity levels during acute and chronic inflammation. (4) The hypothesis that the amino- terminal portion of SAA is required for fibrillogenesis will be tested using variant rSAA proteins. Fibrils formed in vitro from rSAAs will be examined by light and electron microscopy. Accomplishment of these aims will contribute appreciably to a better understanding of SAA metabolism and, in particular, will provide insight into the interrelationships of HDL-binding, degradation, and amyloid fibril formation.